[Show abstract][Hide abstract] ABSTRACT: We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine
infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate
RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added
oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest
that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux
pumps can be used to eliminate non-growing mycobacteria.
[Show abstract][Hide abstract] ABSTRACT: Antimicrobials targeting cell wall biosynthesis are generally considered inactive against non-replicating bacteria. Paradoxically, we found that in non-permissive growth conditions exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, Raas (for regulator of antimicrobial-assisted survival) encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial death in non-permissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination therapies for persistent infectious diseases such as tuberculosis.
[Show abstract][Hide abstract] ABSTRACT: The phosphorylated Spo0A transcription factor controls the initiation of endospore formation in Clostridium acetobutylicum, but genes encoding key phosphorelay components, Spo0F and Spo0B, are missing in the genome. We hypothesized that the five orphan histidine kinases of C. acetobutylicum interact directly with Spo0A to control its phosphorylation state. Sequential targeted gene disruption and gene expression profiling provided evidence for two pathways for Spo0A activation, one dependent on a histidine kinase encoded by cac0323, the other on both histidine kinases encoded by cac0903 and cac3319. Purified Cac0903 and Cac3319 kinases autophosphorylated and transferred phosphoryl groups to Spo0A in vitro, confirming their role in Spo0A activation in vivo. A cac0437 mutant hyper-sporulated, suggesting that Cac0437 is a modulator that prevents sporulation and maintains cellular Spo0A∼P homeostasis during growth. Accordingly, Cac0437 has apparently lost the ability to autophosphorylate in vitro; instead it catalyses the ATP-dependent dephosphorylation of Spo0A∼P releasing inorganic phosphate. Direct phosphorylation of Spo0A by histidine kinases and dephosphorylation by kinase-like proteins may be a common feature of the clostridia that may represent the ancestral state before the great oxygen event some 2.4 billion years ago, after which additional phosphorelay proteins were recruited in the evolutionary lineage that led to the bacilli.
[Show abstract][Hide abstract] ABSTRACT: Micrococcus luteus (NCTC2665, "Fleming strain") has one of the smallest genomes of free-living actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp (G+C content, 73%) predicted to encode 2,403 proteins. The genome shows extensive synteny with that of the closely related organism, Kocuria rhizophila, from which it was taxonomically separated relatively recently. Despite its small size, the genome harbors 73 insertion sequence (IS) elements, almost all of which are closely related to elements found in other actinobacteria. An IS element is inserted into the rrs gene of one of only two rrn operons found in M. luteus. The genome encodes only four sigma factors and 14 response regulators, a finding indicative of adaptation to a rather strict ecological niche (mammalian skin). The high sensitivity of M. luteus to beta-lactam antibiotics may result from the presence of a reduced set of penicillin-binding proteins and the absence of a wblC gene, which plays an important role in the antibiotic resistance in other actinobacteria. Consistent with the restricted range of compounds it can use as a sole source of carbon for energy and growth, M. luteus has a minimal complement of genes concerned with carbohydrate transport and metabolism and its inability to utilize glucose as a sole carbon source may be due to the apparent absence of a gene encoding glucokinase. Uniquely among characterized bacteria, M. luteus appears to be able to metabolize glycogen only via trehalose and to make trehalose only via glycogen. It has very few genes associated with secondary metabolism. In contrast to most other actinobacteria, M. luteus encodes only one resuscitation-promoting factor (Rpf) required for emergence from dormancy, and its complement of other dormancy-related proteins is also much reduced. M. luteus is capable of long-chain alkene biosynthesis, which is of interest for advanced biofuel production; a three-gene cluster essential for this metabolism has been identified in the genome.
Journal of bacteriology 11/2009; 192(3):841-60. DOI:10.1128/JB.01254-09 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis contains five genes, rpfA through rpfE, that bear significant homology to the resuscitation-promoting factor (rpf) gene of Micrococcus luteus, whose product is required to resuscitate the growth of dormant cultures of M. luteus and is essential for the growth of this organism. Previous studies have shown that deletion of any one of the five rpf-like genes did not affect the growth or survival of M. tuberculosis in vitro. In conjunction with the results of whole-genome expression profiling, this finding was indicative of their functional redundancy. In this study, we demonstrate that the single deletion mutants are phenotypically similar to wild-type M. tuberculosis H37Rv in vivo. The deletion of individual rpf-like genes had no discernible effect on the growth or long-term survival of M. tuberculosis in liquid culture, and the ability to resuscitate spontaneously from a nonculturable state in a most probable number assay was also unaffected for the three strains tested (the DeltarpfB, DeltarpfD, and DeltarpfE strains). In contrast, two multiple strains, KDT8 (DeltarpfA-mutation DeltarpfC DeltarpfB) and KDT9 (DeltarpfA DeltarpfC DeltarpfD), which lack three of the five rpf-like genes, were significantly yet differentially attenuated in a mouse infection model. These mutants were also unable to resuscitate spontaneously in vitro, demonstrating the importance of the Rpf-like proteins of M. tuberculosis in resuscitation from the nonculturable state. These results strongly suggest that the biological functions of the five rpf-like genes of M. tuberculosis are not wholly redundant and underscore the potential utility of these proteins as targets for therapeutic intervention.
Infection and Immunity 06/2005; 73(5):3038-43. DOI:10.1128/IAI.73.5.3038-3043.2005 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aged, dormant cultures of Mycobacterium tuberculosis can be resuscitated by a secreted, proteinaceous growth factor from Micrococcus luteus, known as resuscitation-promoting factor (Rpf). M. tuberculosis contains five rpf-like genes, rpfA (Rv0867c), rpfB (Rv1009), rpfC (Rv1884c), rpfD (Rv2389c) and rpfE (Rv2450c), that bear significant similarity to Mi. luteus rpf, suggesting that these too may play a role in growth and/or reactivation from a quiescent state.
Unmarked deletion mutants of each of the five rpf-like genes of M. tuberculosis H37Rv were constructed and their phenotypes and global gene expression profiles were assessed.
Deletion of any one of the rpf-like genes did not affect growth or survival of M. tuberculosis in liquid culture, but some alterations in colony-forming ability and colonial morphology were observed. Global gene expression profiling suggested that loss of rpfC affected the expression of the largest number of genes and there was a significant overlap in the differential gene expression profile of the rpfC mutant with those of the rpfB, rpfD and rpfE mutants. The expression profile of the rpfA mutant was notably less similar, but inverse associations with genes affected in the other mutants were observed. These results, together with those obtained by real-time, quantitative RT-PCR, suggest that the rpf-like genes serve wholly or partially overlapping functions in M. tuberculosis.
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus. Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show here that the five cognate proteins from M. tuberculosis have very similar characteristics and properties to those of Rpf. They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations. Several lines of evidence indicate that they exert their activity from an extra-cytoplasmic location, suggesting that they are also involved in intercellular signalling. The five M. tuberculosis proteins show cross-species activity against M. luteus, Mycobacterium smegmatis and M. bovis (BCG). Actively growing cells of M. bovis (BCG) do not respond to these proteins, whereas bacteria exposed to a prolonged stationary phase do. Affinity-purified antibodies inhibit bacterial growth in vitro, suggesting that sequestration of these proteins at the cell surface might provide a means to limit or even prevent bacterial multiplication in vivo. The Rpf family of bacterial growth factors may therefore provide novel opportunities for preventing and controlling mycobacterial infections.
[Show abstract][Hide abstract] ABSTRACT: Although much is known about the bacterial cellulosome and its various protein components, their contributions to bacterial growth on cellulose and the process of cellulolysis in vivo cannot currently be assessed. To remedy this, the authors have developed gene transfer techniques for Clostridium cellulolyticum ATCC 35319. Firstly, transfer of Tn1545 has been obtained using an Enterococcus faecalis donor. Secondly, IncP-mediated conjugative mobilization of plasmids from Escherichia coli donors has also been achieved. The yield of transconjugants in both cases was low and was probably limited by the suboptimal growth conditions that must of necessity be employed for the co-culture of oligotrophic C. cellulolyticum with copiotrophic donors. A restriction endonuclease was detected in crude extracts of C. cellulolyticum. This enzyme, named CCE:I, is an isoschizomer of MSP:I (HPA:II). Electro-transformation was employed to establish plasmids containing the replication functions of pAMss1 (En. faecalis), pIM13 (Bacillus subtilis), pCB102 (Clostridium butyricum), pIP404 (Clostridium perfringens) and pWV01 (Lactococcus lactis subsp. cremoris) in C. cellulolyticum. Transformants were only obtained if the DNA was appropriately methylated on the external C of the sequence 5'-CCGG-3' using either BSU:FI methylase in vivo or MSP:I methylase in vitro. Plasmids based on the pAMss1 and pIM13 replicons were more stably maintained than one based on the pCB102 replicon. Selection of transformants on solid medium led to low apparent transformation efficiencies (approx. 10(2) transformants per microg DNA) which might, in part, reflect the low plating efficiency of the organism. Selection of transformants in liquid medium led to a higher apparent yield of transformants (between 10(5) and 10(7) transformants per microg DNA). The methods developed here will pave the way for functional analysis of the various cellulosome components in vivo.
[Show abstract][Hide abstract] ABSTRACT: The spo0A genes of Clostridium beijerinckii NCIMB 8052 and Clostridium cellulolyticum ATCC 35319 were isolated and characterized. The C-terminal DNA-binding domains of the predicted products of spo0A from these two organisms, as well as 16 other taxonomically diverse species of Bacillus and Clostridium, show extensive amino acid sequence conservation (56% identity, 65% similarity over 104 residues). A 12-amino-acid motif (SRVERAIRHAIE) that forms the putative DNA recognition helix is particularly highly conserved, suggesting a common DNA target. Insertional inactivation of spo0A in C. beijerinckii blocked the formation of solvents (as well as spores and granulose). Sequences resembling Spo0A-binding motifs (TGNCGAA) are found in the promoter regions of several of the genes whose expression is modulated at the onset of solventogenesis in Clostridium acetobutylicum and C. beijerinckii. These include the upregulated adc gene, encoding acetoacetate decarboxylase (EC 18.104.22.168), and the downregulated ptb gene, encoding phosphotransbutyrylase (EC 2.3.1.c). In vitro gel retardation experiments using C. acetobutylicum adc and C. beijerinckii ptb promoter fragments and recombinant Bacillus subtilis and C. beijerinckii Spo0A suggested that adc and ptb are directly controlled by Spo0A. The binding affinity was reduced when the 0A boxes were destroyed, and enhanced when they were modified to conform precisely to the consensus sequence. In vivo analysis of wild-type and mutagenized promoters transcriptionally fused to the gusA reporter gene in C. beijerinckii validated this hypothesis. Post-exponential phase expression from the mutagenized adc promoter was substantially reduced, whereas expression from the mutagenized ptb promoter was not shut down at the end of exponential growth.
[Show abstract][Hide abstract] ABSTRACT: A physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome has been constructed, encompassing about 90 rare restriction sites. The 14 rrn operons together with about 40 genes have been assigned positions on the map. Genetic analysis and gene transfer have been developed in this organism to enable in vivo analysis of the roles of cloned genes using marker replacement technology. Experiments using the available genetic tools have shown that spo0A plays a cardinal role in controlling several aspects of the transition from exponential growth to stationary phase in C. beijerinckii. These include initiation of sporulation, accumulation of the storage polysaccharide, granulose, and production of acetone and butanol. Several C. beijerinckii and C. acetobutylicum genes concerned with fermentative metabolism, whose expression is modulated at the onset of solventogenesis, contain sequence motifs resembling 0A boxes in their 5' regulatory regions. This invites the speculation that they are under the direct control of Spo0A, and additional data are now required to test this prediction.